Protease inhibitors from marine sponge and sponge-associated microorganisms

Protease inhibitors are proteins or peptides inhibiting the activity of protease and constitute a very important mechanism for regulating protease activity. So far, protease inhibitors have been used for the study of enzyme structures and reaction mechanisms, but recently they have also been used in pharmaceutical, agricultural and industrial fields.

Vietnam Journal of Science and Technology 56 (4) (2018) 405-423 DOI: 10.15625/2525-2518/56/4/10911 Mini-Review PROTEASE INHIBITORS FROM MARINE SPONGE AND SPONGE-ASSOCIATED MICROORGANISMS Tran Thi Hong1, Ton That Huu Dat1, *, Pham Viet Cuong1, Nguyen Thi Kim Cuc2 Mientrung Institute for Scientific Research, VAST, 321 Huynh Thuc Khang Street, Hue city Marine Biochemistry Institute, VAST, 18 Hoang Quoc Viet, Cau Giay, Ha Noi * Email: huudat96@gmail.com Received: 27 November 2017; Accepted for publication: July 2018 Abstract Protease inhibitors are proteins or peptides inhibiting the activity of protease and constitute a very important mechanism for regulating protease activity So far, protease inhibitors have been used for the study of enzyme structures and reaction mechanisms, but recently they have also been used in pharmaceutical, agricultural and industrial fields Compared to terrestrial counterparts, marine environment possesses their unique characters, therefore, they are capable of producing a wide range of novel bioactive compounds including protease inhibitors In our review, a brief overview of protease inhibitors (e.g., classification, mechanisms, and characteristics of protease inhibitor) and protease inhibitors from marine sponges and sponge-associated microorganisms has been reviewed Keywords: action mechanisms, marine sponge, protease, protease inhibitors, sponge-associated microorganisms Classification numbers: 1.2.1; 1.5.1; 1.5.3 INTRODUCTION OF PROTEASE INHIBITOR Proteases, also called proteolytic enzymes, are enzymes capable of hydrolyzing peptide bonds of peptide chains and proteins into shorter peptides and amino acids [1] They are responsible for different physiological functions in the body such as activating zymogene, clotting and degrading fibrin fiber of blood clots, releasing hormones and biologically active peptides from precursors as well as transporting protein across the membrane Although proteases play a crucial role in host cells, they can be harmful in excess or high concentrations They can activate cancer and cause many diseases (e.g., neurological disorders, inflammation, and cardiovascular diseases) [1, 2] Therefore, the function of these proteolytic enzymes should be monitored and controlled strictly The most important control systems of protease are protease inhibitors (PIs) These inhibition molecules can block the activity of proteases Furthermore, PIs are also found to have other functions such as activating growth factors, eliminating receptors or promoting cancer [1] Tran Thi Hong, Ton That Huu Dat, Pham Viet Cuong, Nguyen Thi Kim Cuc Natural inhibitors were first reported in 1894 by Femi and Pernossi when they discovered antisera activity in human serum [3] To date, PIs have been found extensively in nature from different sources including animals, plants, and microorganisms (Table 1) Table Several protease inhibitors from animal, plant, and bacterial sources [4, 5] Inhibitors Source Inhibited protease Ov-SPI-l Anisakis simplex inhibitor A PIs from animals Onchocerca volvulus Anisakis simplex AceKI Ancylostoma ceylanicum Chagasin SGTI RsTI CVPI CGPI PSKP & LTI Trypanosoma cruzi Schistocerca gregaria Rhipicephalus sanguineus Crassostrea virginica Crassotrea gigas Phyllomedusa sauvagii Lymnaea Serine Serine Trypsin, Chymotrypsin, Pancreatic elastase Endogenous cysteine Bovine trypsin Trypsin protease Thermolysin Thermolysin Endo peptidase Trypsin B PIs from plants Glycine max Hordeum vulgare Trypsin, Chymotrypsin Subtilisin, α-amylase Psophocarpus tetragonolobus α-chymotrypsin Solanum tuberosum Cysteine proteases Kunitz trypsin inhibitor Barley subtilisin inhibitor Chymotrypsin Inhibitor Kunitz cysteine peptidase inhibitor Proteinase inhibitor A inhibitor unit Kunitz subtilisin inhibitor Canavalia lineata Cathepsin D inhibitor Trypsin inhibitor Ragi seed trypsin/α-amylase inhibitor Barley trypsin/factor XIIa inhibitor Trypsin/α-amylase inhibitor Trypsin/factor XIIa inhibitor Trypsin inhibitor MCTI-1 Trypsin inhibitor MCTI-II Macrocyclic squash trypsin inhibitor Solanum tuberosum Acacia confusa Trypsin, Chymotrypsin, Kallikerin Subtilisin-type microbial serine proteases Cathepsin D, Trypsin Trypsin, α-chymotrypsin Eleusine coracana α-amylase Hordeum vulgare α-amylase, Trypsin Triticum aestivum α-amylase, Trypsin 406 Sagittaria sagittifolia Momordica charantia Momordica charantia Mammalian trypsin, activated hageman factor Pancreatic elastase Trypsin Momordica cochinchinensis Trypsin Zea mays Protease inhibitors from marine sponge and sponge-associated microorganisms Trypsin inhibitor CSTI-IV Chymotrypsin inhibitor I Cucumis sativus Solanum tuberosum Glutamyl peptidase II Momordica charantia Subtilisin-chymotrypsin inhibitor CI-1A Subtilisin/chymotrypsin inhibitor Mustard trypsin inhibitor Hordeum vulgare Trypsin Chymotrypsin, Trypsin Glu S griseus protease , Subtilisin Subtilisin, Chymotrypsin Mustard trypsin inhibitor-2 Brassica hirta Rape trypsin inhibitor Metalloprotease inhibitor Sarcocystatin Bowman–Birk plant trypsin inhibitor unit Bowman-Birk trypsin/ chymotrypsin inhibitor Brassica napus Bothrops jararaca Sarcophaga peregrina B lichenoformis subtilisin, α-chymotrypsin Beta-trypsin Bovine beta-trypsin, α-Chymotrypsin Trypsin, Chymotrypsin Atrolysin C, Jararhagin Cysteine proteinase Glycine max Trypsin, Chymotrypsin Arachis hypogaea Trypsin, Chymotrypsin Sunflower cyclic trypsin inhibitor Proteinase inhibitor II Potato peptidase inhibitor II inhibitor unit Tomato peptidase inhibitor II inhibitor unit Tomato peptidase inhibitor II inhibitor unit Marinostatins Monastatin POI Lentinus proteinase SLPI SMPI Sma PI Triticum aestivum Sinapis alba Solanum tuberosum Trypsin, Cathepsin G, Elastase, Chymotrypsin and thrombin Trypsin, Chymotrypsin Solanum tuberosum Trypsin, Chymotrypsin Solanum lycopersicum Trypsin, Chymotrypsin Solanum lycopersicum Trypsin, Chymotrypsin C PIs from microorganisms Alteromonas sp Alteromonas sp Pleurotus ostreatus Lentinus edodes Streptomyces lividans Streptomyces nigrescens Serratia marcescens Cysteine Serine Serine proteinase A Trypsin Subtilisin BPN Metalloprotease Metalloprotease Helianthus annuus PIs are known as one of the important catalysts in protein purification procedures as they may minimize proteolysis during heterologous expression or protein extraction Additionally, PIs may support for effective purification of proteases using affinity chromatography In medicine, PIs can be used for diagnosing and treating different diseases (e.g., viral, bacterial, fungal and parasitic diseases, cancer and immunological, neurodegenerative and cardiovascular diseases [6] In some circumstances, PIs may be used as drugs for the treatment of diseases using the synthetic inhibitors or the natural inhibitors [7, 8] Approximately, 32 PIs are currently in clinical use, most of them are synthetic molecules developed by structure-based design [9] In addition, several protease inhibitors found in natural sources are also in clinical use For examples, an aspartic protease inhibitor of HIV-1 (ritonavir) has been used since 1996 for the 407 Tran Thi Hong, Ton That Huu Dat, Pham Viet Cuong, Nguyen Thi Kim Cuc AIDS treatment, and boceprevir and telaprevir also approved by the FDA in 2011 for the treatment of hepatitis C virus infection [10] Furthermore, protease inhibitors can be involved in crop protection against plant pathogens and herbivorous pests in agriculture [11] Exploration and use of novel PIs with protective function are one of the important tools in crop protection and the development of environmentally friendly pest and pathogen management strategies The genetically modified plants expressing inhibitors of the digestive enzymes of their insect pests are already under study [12, 13] CLASSIFICATION OF PROTEASE INHIBITORS PIs may be classified based dimensional), the source organism (broad range, specific), and action reversible or irreversible) or based serine protease inhibitors) on different ways: their structure (primary and three(microbial, fungal, plant, animal), their inhibitory profile mechanism (competitive, non-competitive, uncompetitive, on the class of protease they inhibit (aspartic, cysteine or Currently, PIs are commonly grouped into two groups: (1) small molecule inhibitors and (2) proteinaceous inhibitors Small molecule inhibitors (SMIs) SMIs are low molecular mass peptides and synthetic inhibitors from microorganisms They are inhibitors that are not proteins, include natural compounds (e.g., pepstatin, bestatin and amastatin) as well as synthetic inhibitors generated in a laboratory [14] To date, most of the natural SMIs have been isolated from bacteria and fungi [15] Each SMI is named by an initial J followed by a five-digit number For example, pepstatin is J00095 [16] Several SMIs have proved useful in inhibition of diseases such as retropepsin of the HIV virus [17], thrombin of thrombosis, dipeptidyl-peptidase IV (18, 19, 20], γ-secretase of Alzheimer’s disease [21], renin [22] and angiotensin-converting enzyme of blood pressure [23], and peptidases from the malarial parasite Plasmodium [24] Furthermore, some SMIs are also found as anticancer and antinutritional agents [20, 25, 26, 27, 28] Proteinaceous inhibitors Proteinaceous inhibitors are ubiquitous inhibitors and isolated from different sources (e.g., microorganisms, plants, and animals) Natural proteinaceous inhibitors are known as templates for the modification of natural control mechanisms and as a source of basic design principles [29] Proteinaceous inhibitors are usually classified based on the kind of inhibited protease Currently, hundreds of protein inhibitors of peptidases are known [30] According to lastest update of MEROPS database (http://www.ebi.ac.uk/merops/), PIs are grouped into 83 families based on comparisons of protein sequences However, molecular weight and mechanism of inhibition of PIs are dissimilar Therefore, these families are further grouped into clans based on comparisons of their tertiary structure Each clan, family and biochemically characterized peptidase inhibitor is assigned a unique identifier A family is identified by a letter “I” followed by a number and two-letter clan identifier starts with “I” or “J” [14, 30] Some families of proteinaceous inhibitors from microbes and fungi are listed in Table 408 Protease inhibitors from marine sponge and sponge-associated microorganisms MECHANISM OF PROTEASE INHIBITORS Competitive inhibition The majority of PIs are known as competitive inhibitors Generally, these inhibitors often bind to the active sites of target proteases in a substrate-like manner (Figure 1) In some case, the competitive inhibitors bind in and block access to the active site of target proteases, but not bind in a strictly substrate-like manner They may interact with protease subsites and catalytic residues in a non-catalytically competent manner [31] Although the competitive mechanism is considered as an effective strategy of competitive inhibitors, the proteases often have a high degree of homology in the active sites, substrate-like binding may, therefore, lead to inhibitors that can inhibit many different proteases [31] For example, the activity of 612 known human proteases is regulated by about 115 human protease inhibitors [9] The inhibitors of serine protease including the Kazal, Kunitz, and Bowman-Birk family are examples of competitive inhibitors [32] Table Families of proteinaceous inhibitors of microbial and fungal origin [29] Family I1 I2 I4 I9 I10 I11 I16 I31 I32 I34 I36 I38 Common name Kazal Kunitz-BPTI Serpin YIB Marinostatin Ecotin SSI Thyropin IAP IA3 SMI Aprin 139 - I42 I43 I48 I51 I57 I58 I63 I66 I69 I75 I78 I79 I85 I87 Chagasin Oprin Clitocypin IC Staphostatin B Staphostatin A Cnispin CIII AVR2 Macrocypin Hf1KC Families of peptidases inhibited M10, S1A, S1D, S8A, S9A S1A, S7 C1A, C14A, S1A, S7, S8A, S8B S8A S1A, S8A S1A M4, M7, S1A, S8A, S8B A1A, C1A, M10A C14A A1A M4 M10B A1A, A2A, C1A, C2A, C11, M4, M10A, M10B, M12A, M12B, S1A, S1B, S8A C1A M12B C1A, C13 S1A, S10 C47 C47 M43B, S1A S1A C10 M41 S1A, S8A C1A C1A, C13, S1A M41 409 Tran Thi Hong, Ton That Huu Dat, Pham Viet Cuong, Nguyen Thi Kim Cuc Figure Competitive inhibitors of proteases (A) Inhibitors bind in the active site, but not in a substratelike manner Peptide extensions bind in specificity subsites and can interact with the catalytic residues (rectangle) (B) Crystal structures of a serine protease in complex with the standard mechanism inhibitor aprotinin, and (C) the cystatin stefin A in complex with a cysteine protease The portion of stefin A that interacts with the protease is coloured in green Both inhibitors bind in the active site groove of their targets [31] The reuse of this figure was permitted by John Wiley and Sons publisher under licensed number: 4390911096682 Figure The competitive inhibitors with exosite binding (A) Most exosite inhibitors are competitive inhibitors that prevent substrate binding at the active site In the case of (B) ecotin (bound to trypsin), the exosites provide binding energy and allow for broad specificity, while (C) calpastatin gains binding energy and specificity by forming critical interactions across the calpain protease surface [31] The reuse of this figure was permitted by John Wiley and Sons publisher under licensed number: 4390911096682 Competitive inhibition with exosite binding In some PIs, they are not only competitive and bind to the protease active site, but also bind to secondary sites outside the active site [31] This inhibition mechanism has two benefits Firstly, it may increase the surface area of the protein-protein interaction, which results in a greater affinity Secondly, it can provide a significant effect on the specificity of the inhibitor 410 Protease inhibitors from marine sponge and sponge-associated microorganisms [31] Some inhibitors such as Rhondiin, Ecotin, and Calpastatin are examples of this inhibition mechanism (Figure 2) Irreversible inhibition Generally, irreversible inhibitors activate the proteolysis by the enzymes they inhibit, leading to a covalent modification of the enzyme In this mode, inhibitors act as substrates in order to trap and inhibit the enzyme using the enzyme’s catalytic machinery [31] The serpins, a family of inhibitors act as the irreversible inhibitors (Figure 3) Figure Serpins inhibit serine proteases by binding a reactive centre loop in the active site, forming a covalent complex with the enzyme, undergoing a large conformational change, and irreversibly distorting the active site of the protease [31] The reuse of this figure was permitted by John Wiley and Sons publisher under licensed number: 4390911096682 CHARACTERIZATION OF PROTEASE NHIBITORS PIs are one of the most important class of proteins that can be applied to various fields Therefore, characterizing PIs is crucial for determining their scope and application The determination of the physicochemical properties and the structural stability of PIs is key to select effective and stable inhibitors for their application pH and temperature stability Thermal stability of PIs is one of the important properties for their biotechnological applications The extreme conditions of pH and temperature might cause the distortion of the structure of the PIs Therefore, the combination of inhibitors with the enzymes or their substrates may be broken [33] Ryan et al [34] reported that many PIs exhibiting anti-feedant activity are active against the neutral serine proteases such chymotrypsin and trypsin The pH and temperature stability of the PIs can also be involved in the presence of disulfide linkages [35] The functional stability of Kunitz type PIs are related to the intra-molecular disulphide bridges in the presence of physical and chemical denaturants (e.g., temperature, pH and reducing agents) [36] Effect of metal ions The presence of metal ions is essential for the activity of PIs The metal ions play an important role in maintaining the structure of PIs The structural stability of proteins is enhanced by divalent metal ions as the metal ions can attain the critical conformation that is needed for 411 Tran Thi Hong, Ton That Huu Dat, Pham Viet Cuong, Nguyen Thi Kim Cuc biological activity of the protein [29] For example, cysteine protease inhibitor from pearl millet needs the Zn2+ for the protease inhibitory and antifungal activity of the protein [37] Effect of oxidizing agents The proteins may be oxidized when they expose to oxidising agents (e.g., H2O2, periodate, dimethyl sulfoxide, N-chlorosuccinamide, chloramine-T), and to oxidants released by neutrophils (e.g superoxide, hydroxyl radical) [38, 39] The oxidation of methionine residues may result in a decline in the biological activity of the protein A report of the influence of methionine oxidation on α1-protease inhibitor (α1-PI) showed that the oxidation of one of the methionine residues (Met358) lead to a complete loss of inhibitory activity of the α1-protease inhibitor [40] Effect of reducing agents In some proteins, the covalent linkage of cysteine residues by disulfide bonds is one of the crucial elements in maintaining their conformational stability and biological activity This covalent linkage plays a crucial role in the proper folding, stability and function of many proteins [41, 42, 43] Conformational destabilization of the protein may result from the removal of the covalent link of cysteine residues caused by reduction or substitution of another amino acid residue [44, 45] Effect of detergents Some detergents (e.g., cationic, anionic, zwitterionic and non-ionic) are used for solubilizing proteins from lipid membranes Therefore, proteinase inhibitors are often combined with detergents in cell lysis buffers to inhibit undesired proteolysis and facilitate membrane protein solubilization in protein purification procedures [29] Normally, nonionic detergents are considered as mild detergents and not interact extensively with the protein surface, while ionic detergents (e.g., SDS) generally bind unwanted to the protein surface, which results in protein unfolding [46] Additionally, Triton X-100, Tween 20 and Tween 80 are nonionic detergents and the majority of their interaction with proteins are hydrophobic [47] Effect of chemical modifiers Chemical modification is a useful method for changing undesired characteristics of a protein related to stability and catalytic activity In a chemical modification, the chemical reagents bind covalently to specific amino acid chains of proteins and produce changes in the biological property of the protein [48, 49] For example, Urwin et al (1995) reported that chemical modification might enhance the activity of PIs against proteinases of the pests [50] In other studies, chemical modification of soybean cystatin scN and tomato multicystatin reveals the considerable influences of the substitution of individual amino acid residues in the Nterminal portion of one of multicystatin domains on its ability to inhibit diverse proteinases [51, 52] PROTEASE INHIBITORS FROM SPONGE AND SPONG-ASSOCIATED MICROORGANISMS Protease inhibitor from marine sponges Although marine sponges are known as the most simple organisms, they are able to produce a great number of biologically active compounds, including PIs A summary table of compounds from marine sponges with protease inhibitory activity is shown in Table The compounds with protease inhibitory activity from marine sponge are diverse and exhibit inhibitory activity 412 Protease inhibitors from marine sponge and sponge-associated microorganisms against many different proteases For example, cyclotheonellazoles isolated from sponge Theonella inhibited various proteases such as chymotrysin, elastase, malaria parasite from Plasmodium falciparum, thrombin, plasmin [53] Cyclotheonamides from sponge Theonella swinhoei and Theonella sp also inhibited two proteases thrombin (IC50 = 5.2 - 13 nM) and trypsin (IC50 = 7.4 - 370 nM) [54, 55] Interestingly, other compounds from the same sponge species Theonella swinhoei such as nazumazoles, pseudotheonamides, dihydrocyclotheonamide A showed inhibitory activity against proteases (e.g., thrombin, RCE-protease, chymotrypsin) [65, 70] To date, many new compounds extracted from various sponge have been known as protease inhibitors (see Table 3), indicating that marine sponge is one of the potential sources for mining protease inhibitors Table 3: Protease inhibitors from marine sponges Compounds Sponge Inhibited protease and activity Ref Cyclotheonellazole A Theonella aff swinhoei Cyclotheonellazole B Theonella aff swinhoei Cyclotheonellazole C Theonella aff swinhoei Cyclotheonamide A Theonella sp Cyclotheonamide C Theonella swinhoei Cyclotheonamide D Theonella swinhoei Cyclotheonamide E Theonella swinhoei Cyclotheonamide E2 Theonella sp Cyclotheonamide E3 Theonella sp Plakortide E Plakortis halichondroides Miraziridine A Theonella swinhoei Chymotrypsin (IC50 = 0.62 nM) Elastase (IC50 = 0.034 nM) Malaria parasite (IC50 > 20 µg/mL) Chymotrypsin (IC50 = 2.8 nM) Elastase (IC50 = 0.10 nM) Malaria parasite (IC50 > 20 µg/mL) Chymotrypsin (IC50 = 2.3 nM) Elastase (IC50 = 0.099nM) Malaria parasite (IC50 > 20 µg/mL) Thrombin (IC50 = 0.076 µg/mL) Trypsin (IC50 = 0.2 µg/mL) Plasmin (IC50 = 0.3 µg/mL) Thrombin (IC50 = 8.4 nM) Trypsin (IC50 = 7.4 nM) Thrombin (IC50 = 5.2 nM) Trypsin (IC50 = 63 nM) Thrombin (IC50 = 28 nM) Trypsin (IC50 = 370 nM) Thrombin (IC50 = 13 nM) Trypsin (IC50 = 55 nM) Thrombin (IC50 = 9.5 nM) Trypsin (IC50 = 52 nM) Cathepsin B, cathepsin L, falcipain, rhodesain, SARS Mpro, SARS PLpro, DENV-2pro, Chymotrypsin (inhibition 10 – 90%) Cathepsin L (inhibition 60%) Miraziridine A Theonella aff mirabilis Trypsin, cathespin L, cathespin B, pepsin [59] Tokaramide A 1-methylherbipoline salts of halisulfate-1 1-methylherbipoline salts of sulvanine Theonella aff mirabilis [60] Sodium salt of halisulfate-1 Coscinoderma mathewsi Sodium salts of suvanine Coscinoderma mathewsi Cathepsin B (IC50 = 29.0 µg/mL) Thrombin( IC50 > 100 µg/mL) Trypsin (IC50 = 25 µg/mL) Thrombin (IC50 = 27 µg/mL) Trypsin (IC50 = 12 µg/mL) Thrombin (IC50 = 35 µg/mL) Trypsin (IC50 = µg/mL) Thrombin (IC50 = µg/mL) Trypsin (IC50 = 27 µg/mL) Coscinoderma mathewsi Coscinoderma mathewsi [53] [53] [53] [54] [55] [55] [55] [56] [56] [57] [58] [61] [61] [61] [61] 413 Tran Thi Hong, Ton That Huu Dat, Pham Viet Cuong, Nguyen Thi Kim Cuc N,N-dimethylguanidium salts of suvanine Coscinoderma mathewsi Dysinosin A Lamellodysidea chlorea Dysinosin B Lamellodysidea chlorea Dysinosin C Lamellodysidea chlorea Dysinosin D Lamellodysidea chlorea Crude extracts Jaspis stellifera Crude extracts Esculetin-4-carboxylic acid ethyl ester Plakortis nigra Thrombin (IC50 = 25 µg/mL) Trypsin (IC50 = 23 µg/mL) Thrombin (Ki = 0.108 µM) FVIIa (Ki = 00.452 µM) Thrombin (Ki = 0.090 µM) FVIIa (Ki = 0.170 µM) Thrombin (Ki = 0.124 µM) FVIIa (Ki = 0.550 µM) Thrombin (Ki = 1.320 µM) FVIIa (Ki > 5.1 µM) E coli protease (MIC = 0.08%) S aureus protease (MIC = 0.08%) S aureus protease (MIC = 0.12%) Axinella cf corrugata SARS-coronovirus 3CL (ID50 = 46mM/L) Pseudotheonamide A1 Theonella swinhoei Pseudotheonamide A2 Theonella swinhoei Pseudotheonamide B2 Theonella swinhoei Pseudotheonamide C Theonella swinhoei Pseudotheonamide D Theonella swinhoei Dihydrocyclotheonamide A Theonella swinhoei Barangcadoic acid A [61] [62] [62] [62] [62] [63] [63] [64] Hippospongia sp Thrombin (IC50 = 1.0 µM) Trypsin (IC50 = 4.5 µM) Thrombin (IC50 = 3.0 µM) Trypsin (IC50 > 10 µM) Thrombin (IC50 = 1.3 µM) Trypsin (IC50 = 6.2 µM) Thrombin (IC50 = 0.19 µM) Trypsin (IC50 = 3.8 µM) Thrombin (IC50 = 1.4 µM) Trypsin (IC50 > 10 µM) Thrombin (IC50 = 0.33 µM) Trypsin (IC50 = 6.7 µM) RCE-protease (IC50 = 10 µg/mL) [66] Rhopaloic acid A Hippospongia sp RCE-protease (IC50 = 10 µg/mL) [66] Rhopaloic acid B Hippospongia sp RCE-protease (IC50 = 10 µg/mL) [66] Rhopaloic acid C Hippospongia sp RCE-protease (IC50 = 10 µg/mL) [66] Rhopaloic acid D Hippospongia sp RCE-protease (IC50 = 10 µg/mL) [66] Rhopaloic acid E Hippospongia sp RCE-protease (IC50 = 10 µg/mL) [66] Crude extract C-29EA Amphimedon sp NS3 protease (IC50 = 10.9 µg/mL) [67] Toxadocial A 5,9,23-Triacontatrienoic methyl ester Nazumazole D Toxadocia cylindrical Thrombin (IC50 = 6.5 µg/mL) [68] Chondrilla nucula Elastase (ID50 = 10 µg/mL) [69] Theonella swinhoei Chymotrypsin (IC50 = µM) [70] Nazumazole E Theonella swinhoei Chymotrypsin (IC50 = µM) [70] Nazumazole F Theonella swinhoei Chymotrypsin (IC50 = 10 µM) [70] Asteropterin Asteropus simplex Cathepsin B (IC50 = 1.4 µg/mL) [71] Shishicrellastatin A Crella (Yvesia) spinulata Cathepsin B (IC50 = µg/Ml) [72] Shishicrellastatin B Crella (Yvesia) spinulata Cathepsin B (IC50 = µg/mL) [72] Xestosaprol F Xestospongia sp BACE1 (IC50 = 135 µM) [73] 414 [65] [65] [65] [65] [65] [65] Protease inhibitors from marine sponge and sponge-associated microorganisms Xestosaprol G Xestospongia sp BACE1 (IC50 = 155 µM) [73] Xestosaprol H Xestospongia sp BACE1 (IC50 = 82 µM) [73] Xestosaprol I Xestospongia sp BACE1 (IC50 = 163 µM) [73] Xestosaprol J Xestospongia sp BACE1 (IC50 = 90 µM) [73] Xestosaprol K Xestospongia sp BACE1 (IC50 = 93 µM) [73] Xestosaprol L Xestospongia sp BACE1 (IC50 = 98 µM) [73] Xestosaprol M Xestospongia sp [73] Ancorinoside B Penares sollasi Ancorinoside C Penares sollasi BACE1 (IC50 = 104 µM) MT1-MMP (IC50 = 500 µg/mL) MMP2 (IC50 = 33 µg/mL) MT1-MMP (IC50 = 370 µg/mL) [74] Ancorinoside D Penares sollasi MT1-MMP (IC50 = 180 µg/mL) [74] Ancorinoside A Penares sollasi [74] Ageladine A Agelas nakamurai MT1-MMP (IC50 = 440 µg/mL) MMP-1 (IC50 = 1.2 µg/mL) MMP-2 (IC50 = 2.0 µg/mL) MMP-8 (IC50 = 0.39 µg/mL) MMP-9 (IC50 = 0.79 µg/mL) MMP-12 (IC50 = 0.33 µg/mL) MMP-13 (IC50 = 0.47 µg/mL) mammalian aminopeptidase N [76] MMP-2 [77] Psammaplin A Aeroplysinin-1 Poecillastra sp Jaspis sp Marine sponge [74] [75] Protease inhibitor from sponge-associated microorganisms Marine sponges are one of the most potential producers of bioactive agents among marine organisms They have been proven to be a rich source of novel secondary metabolites with diverse bioactive activities (e.g., anticancer, antibiotic, protease inhibitory activity) [78, 79, 80, 81, 82] However, there is still an ongoing debate about whether known bioactive compounds from sponges are originated from sponges or from their associated symbionts Recent studies have evidenced that many previous compounds isolated from sponges are from their associated microorganisms [83, 84] Although PIs can be found from different sources (e.g., microorganisms, plants, animals), there are a few number studies of PIs from the marine environment, especially from spongeassociated microorganisms Recent studies have shown the potential protease inhibitors isolated from sponge-associated microorganisms (Table 4) The crude extracts from bacteria associated with Caribbean sponges exhibited inhibitory activity against different proteases such as cathepsin B, rhodesain, falcipain-2 In addition, these crude extracts showed immunomodulatory activity via induction of cytokine release by human peripheral blood mononuclear cells [85] In another study, teromycins extracted from Streptomyces axinellae associated with sponge Axinellae polypoides also inhibited various proteases such as rhodesain, falcipain-2, cathepsin-L, cathepsin-B, SARS-CoV-PLpro [86] Furthermore, the crude extracts from bacteria associated with other sponge species (e.g., Jasis sp., Plakortis nigra, Jasis stellifera, Xestospongia testudinaria, Aplysina aerophoba) showed protease inhibitory activity against subtilisin, thermolysin as well as proteases from Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa [87, 88, 89] 415 Tran Thi Hong, Ton That Huu Dat, Pham Viet Cuong, Nguyen Thi Kim Cuc Table 4: Protease inhibitors from sponge-associated microorganisms Compounds Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Crude extract Tetromycin B Tetromycin Sponge Caribbean Sponges Caribbean Sponges Caribbean Sponges Caribbean Sponges Caribbean Sponges Caribbean Sponges Caribbean Sponges Caribbean Sponges Axinella polypoides Axinella polypoides Bacteria Nocardioides sp Rhodesain (inhibition 40 ± %) Agrococcus jenensis Cathepsin B (inhibition 41 ± %) Falcipain-2 (inhibition 44 ± %) Micromonospora coxensis Saccharopolyspora shandongensis Micromonospora coxensis Cathepsin B (inhibition 45 ± %) Cathepsin L (inhibition 43 ± %) Falcipain-2 (inhibition 41 ± %) Rhodesain (inhibition 57 ± %) Sphingobium sp Rhodesain (inhibition 53 ± %) Sphingomonas mucosissima Cathepsin B (inhibition 49 ± %) Falcipain-2 (inhibition 45 ± %) Rhodesain (Ki = 0.62 ± 0.03 µM) Falcipain-2 (Ki = 1.42 ± 0.01 µM) Cathepsin L (Ki = 32.5 ± 0.05 µM) Cathepsin B (Ki = 1.59 ± 0.09 µM) SARS-CoV-PLpro (Ki = 69.6 ± 7.2 µM) Rhodesain (Ki = 2.1 ± 0.9 µM) Falcipain-2 (Ki = 1.65 ± 0.25 µM) Cathepsin L (Ki = 15.0 ± 1.95 µM) Cathepsin B (Ki = 0.57 ± 0.04 µM) Rhodesain (Ki = 4.0 ± 0.3 µM) Falcipain-2 (Ki = 3.1 ± 0.2 µM) Cathepsin L (Ki = 22.4 ± 0.8 µM) Cathepsin B (Ki = 1.6 ± 0.1 µM) SARS-CoV-Plpro (Ki = 40 ± 6.5 µM) Rhodesain (Ki = 98 µM) Cathepsin L (IC50 = 72.4 ± 5.3 Μm) Subtilisin (inhibition 91.57 %) Thermolysin (inhibition 59.4 7%) E coli protease (inhibition 98.84 %) Subtilisin (inhibition 57.23 %) Thermolysin (inhibition 70.37 %) S aureus protease (inhibition 51.29 %) Subtilisin (inhibition 30.78 %) Thermolysin (inhibition 50.52 %) P aeruginosa protease (inhibition 23.52 %) P aeruginosa protease (inhibition 72.7 %) Streptomyces axinellae Streptomyces axinellae Diazepinomicin Aplysina aerophoba Micromonospora Crude extract Jaspis sp Providencia sp Crude extract Jaspis sp Crude extract Jaspis sp Crude extract Plakortis nigra 416 Rhodesain (inhibition 52 ± %) Cathepsin L (inhibition 44 ± %) Axinella polypoides Jaspis sp Falcipain-2 (inhibition 42 ± %) Rhodococcus sp Tetromycin Crude extract Inhibited protease and activity Streptomyces axinellae Bacillus sp Paracoccus sp Unidentified bacteria Unidentified bacteria E coli protease (inhibition 93.5 %) Ref [85] [85] [85] [85] [85] [85] [85] [85] [86] [86] [86] [87] [88] [88] [88] [89] [89] Protease inhibitors from marine sponge and sponge-associated microorganisms Crude extract Crude extract Jaspis stellifera Xestospongia testudinaria Unidentified bacteria Chromohalobacter sp S aureus protease (inhibition 40.0 %) P aeruginosa protease (inhibition 95.5 %) [89] [89] In spite of continuous attempts of discovering novel PIs from sponge-associated microorganisms, it is still a big challenge as an only minor fraction of sponge-associated microorganisms can be culture in vitro Fortunately, the new advance approaches (e.g., metagenomics) provide powerful tools for discovering the biosynthetic gene clusters related to polyketide synthases and PIs from uncultured microorganisms [90] This opens the new avenues for detecting novel bioactive metabolites including PIs in future For example, a novel serine protease inhibitor (serpin) gene was detected and cloned from a metagenomic library of uncultured marine microorganisms The phylogenetic analysis and the deduced amino acid sequence comparison of this gene indicated that it was closely related to Spi1C and some partial proteinase inhibitor I4 serpins Furthermore, functional analyses demonstrated that the recombinant Spi1C protein could inhibit a series of serine proteases [91] CONCLUSION In this review, we summarised protease inhibitors with focusing on their classification, action mechanism, and characters as well as protease inhibitors from marine sponge and spongeassociated microorganisms The marine environment poses unique characters and provides a prolific resource for novel bioactive compounds Therefore, continuous efforts in the discovery of structure, functions, biophysical characterization, and mode of action of PIs from marine environment such marine sponge and sponge-associated microorganisms can open up opportunities for their potential role in medicine, biotechnology and agriculture Acknowledgements This research was supported by Ministry of Science and Technology under grant number ĐTĐLCN.17/14 REFERENCES Barrett A J., Rawlings N D., Woessner J J F - Hand book of proteolytic enzymes Academic Press London (1998) Chambers R C., Laurent G J - Coagulation cascade protease and tissue fibrosis, Biochem Soc Trans 30 (2) (2001) 194-200 Fermi C., Pernossi L - Untersuchungen uber die enzyme, Vergleichende Studie, Z Hyg Infektionskr 18 (1) (1894) 83-89 Bijina B - Isolation, purification and characterization of protease inhibitor from Moringa oleifera LAM Ph.D Dissertation, Cochin University of Science and Technology, (2006) Habib H., Fazili K M - Plant protease inhibitors: a defense strategy in plants, Biotechnology and Molecular Biology review (3) (2007) 68-85 Sabotic J., Kos J - Microbial and fungal protease inhibitors-current and potential applications, Appl Microbiol Biotechnol 93 (4) (2012) 1351-1375 Grant P., Mackie A - Drugs from the sea-facts and fantasy, Nature 267 (1977) 786-788 Hamilton S C., Farchaus J W., Davis M C - DNA polymerases as engines for 417 Tran Thi Hong, Ton That Huu Dat, Pham Viet Cuong, Nguyen Thi Kim Cuc biotechnology, Biotechniques 31 (2) (2001), 370-393 Turk B - Targeting proteases: successes, failures and future prospects, Nat Rev Drug Discov (9) (2006) 785–799 10 Fitzpatrick F - Cyclooxygenase enzymes: Regulation and function, Curr Pharma Des 10 (6) (2004) 577-588 11 Sabotič J., Kos J - Microbial and fungal protease inhibitors—current and potential applications, Appl Microbiol Biotechnol 93 (4) (2012) 1351–1375 12 Samac D., Smigocki A - Expression of oryzacystatin I and II in alfalfa increases resistance to the root-lesion nematode, Phytopathology 93 (7) (2003) 799–804 13 Telang M., Patankar A., Harsulkar A., Joshi V., Damle A., Deshpande V., Sainani M., Ranjekar P., Gupta G - Bitter gourd proteinase inhibitors: potential growth inhibitors of Helicoverpa armigera and Spodoptera litura, Phytochemistry 63 (6) (2003) 643–652 14 Umezawa H - Low molecular weight enzyme inhibitors of microbial origin, Ann Rev0 Microbiol 36 (1982) 75-99 15 Rawlings N D - Peptidase inhibitors in the MEROPS database, Biochimie 92 (11) (2010) 1463-1483 16 Rawlings N D., Barrett A J - MEROPS: the peptidase database, Wellcome Trust (2017) 17 Kempf D J., Sham H L., Marsh K C., Flentge C A., Betebenner D., Green B E., Mcdonald E., Vasavanonda S., Saldivar A., Wideburg N E., Kati W M., Ruiz L., Zhao C., Fino L., Patterson J., Molla A., Plattner J J., Norbeck D W - Discovery of ritonavir, a potent inhibitor of HIV protease with high oral bioavailability and clinical efficacy, J Med Chem 41 (4) (1998) 602-617 18 Feng J., Zhang Z., Wallace M B., Stafford J A., Kaldor S W., Kassel D B., Navre M., Shi L., Skene R J., Asakawa T., Takeuchi K., Xu R., Webb D R., Gwaltney S L Discovery of alogliptin: a potent, selective, bioavailable, and efficacious inhibitor of dipeptidyl peptidase IV, J Med Chem 50 (10) (2007)2297-2300 19 Hughes T E., Mone M D., Russell M E., Weldon S C., Villhauer E B - NVPDPP728 (1-[[[2-[(5-cyanopyridin-2-yl) amino]ethyl] amino] acetyl]-2-cyano-(S)-pyrrolidine), a slow-binding inhibitor of dipeptidyl peptidase IV, Biochemistry 38 (36) (1999) 1159711603 20 Kim J H., Mullin C A - Antifeedant effects of proteinase inhibitors on feeding behaviors of adult western corn rootworm (Diabrotica virgifera virgifera), J Chem Ecol 29 (4) (2003) 795–810 21 Imbimbo B P - Alzheimer’s disease: gamma-secretase inhibitors, Drug Discov Today Ther Strat (3) (2008) 169-175 22 Wood J M., Maibaum J., Rahuel J., Grutter M G., Cohen N C., Rasetti V., Ruger H., Goschke R., Stutz S., Fuhrer W., Schilling W., Rigollier P., Yamaguchi Y., Cumin F., Baum H P., Schnell C R., Herold P., Mah R., Jensen C., O’Brien E., Stanton A., Bedigian M P - Structure-based design of aliskiren, a novel orally effective renin inhibitor, Biochem Biophys Res Commun 308 (4) (2003) 698-705 23 Sybertz E J., Watkins R W., Ahn H S., Baum T., Rocca P L., Patrick J., Leitz F Pharmacologic, metabolic, and toxicologic profile of spirapril (SCH 33844),a new angiotensin converting inhibitor, J Cardiovasc Pharmacol 10 (Suppl 7) (1987) S105- 418 Protease inhibitors from marine sponge and sponge-associated microorganisms S108 24 Andrews K T., Fairlie D P., Madala P K., Ray J., Wyatt D M., Hilton P M., Melville L A., Beattie L., Gardiner D L., Reid R C., Stoermer M J., Adams T S., Berry C., McCarthy J S - Potencies of human immunodeficiency virus protease inhibitors in vitro against Plasmodium falciparum and in vivo against murine malaria, Antimicrob Agents Chemother 50 (2) (2006) 639-648 25 Hanada K., Tamai M., Yamagishi M., Ohmura S., Sawada J., Tanaka I - Isolation and characterization of E-64, a new thiol protease inhibitor, Agr Biol Chem Tokyo 42 (3) (1978) 523–528 26 Frlan R., Gobec S - Inhibitors of cathepsin B, Curr Med Chem 13 (19) (2006) 2309– 2327 27 Oppert B., Morgan T D., Kramer K J - Efficacy of Bacillus thuringiensis Cry3Aa protoxin and protease inhibitors against coleopteran storage pests, Pest Manag Sci 67 (5) (2011) 568–573 28 Amirhusin B., Shade R E., Koiw H., Hasegawa P M., Bressan R A., Murdock L L., Zhu-Salzma, K - Protease inhibitors from several classes work synergistically against Callosobruchus maculatus, Journal of Insect Physiology 53 (7) (2007) 734–740 29 Sapna K - Isolation, purification, characterization and application of proteinaceous protease inhibitor from marine bacterium Pseudomonas mendocina BTMW 301 Ph.D Dissertation, Cochin University of Science and Technology, (2006) 30 Rawlings N D., Barrett A J., Thomas P D., Huang X., Bateman A., Finn R D - The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database, Nucleic Acids Research 46 (D1) (2018) D624-D632 31 Farady C J., Craik C S - Mechanisms of macromolecular protease inhibitors, Chembiochem 11 (17) (2010) 2341-2346 32 Laskowski M J., Kato I - Protein inhibitors of proteinases, Ann Rev Biochem 49 (1980) 593-626 33 Veselovsky A V., Ivanov Y D., Ivanov A S., Archakov A I., Lewi P., Janssen, P Protein–protein interactions: mechanisms and modification by drugs, J Mol Recognit 15 (6) (2002) 405–422 34 Ryan C A - Protease inhibitors in plants: genes for improving defenses against insects and pathogens, Ann Rev Phytopatho 28 (1990) 425–449 35 Vernekar J V., Ghatge M S., Deshpande V V - Alkaline protease inhibitor: a novel class of antifungal proteins against phytopathogenic fungi, Biochem Biophys Res Commun 262 (3) (1999) 702−727 36 Kridric M., Fabian H., Brzin J., Popovic T., Pain R - Folding, stability and secondary structure of a new cysteine dimeric proteinase inhibitor Biochem Biophys Res Commun 297 (4) (2002) 962–967 37 Joshi B.N., Sainani M.N., Bastawade K.B., Deshpande V.V., Gupta V.S., Ranjekar P.K Pearl millet cysteine protease inhibitor Evidence for the presence of two distinct sites responsible for anti-fungal and anti-feedant activities, Eur J Biochem., 265 (1999) (2) 556-563 419 Tran Thi Hong, Ton That Huu Dat, Pham Viet Cuong, Nguyen Thi Kim Cuc 38 Brot N., Weissbach H - Biochemistry and physiological role of methionine sulfoxide residues in proteins, Arch Biochem Biophys 223 (1) (1983) 271–281 39 Vogt W - Oxidation of methionyl residues in proteins: tools, targets, and reversal, Free Radical Biol Med 18 (1) (1995) 93-105 40 Johnson D., Travis J - The oxidative inactivation of human alpha-1-proteinase inhibitor Further evidence for methionine at the reactive center, J Biol Chem 254 (10) (1979) 4022-4026 41 Creighton T E., Zapun A., Darby N J - Mechanisms and catalysts of disulfide bond formation in proteins, Trends Biotechnol 13 (1) (1995) 18–23 42 Frand A R., Cuozzo J W., Kaiser C A - Pathways for protein disulphide bond formation, Trends Cell Biol 10 (5) (2000) 203–210 43 Hogg P J - Disulfide bonds as switches for protein function, Trends Biochem Sci 28 (4) (2003) 210–214 44 Kawamura S., Ohkuma M., Chijiiwa Y., Kohno D., Nakagawa H., Hirakawa H., Kuhara S., Torikata T - Role of disulfide bonds in goose-type lysozym, FEBS J 275 (11) (2008) 2818–2830 45 Zavodszky M., Chen C W., Huang J K., Zolkiewski M., Wen L., Krishnamoorthi R Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V, Protein Sci 10 (1) (2001) 149–160 46 Mogensen J E., Sehgal P., Otzen D E - Activation, Inhibition, and destabilization of Thermomyces lanuginosus lipase by detergents, Biochemistry 44 (5) (2005) 1719-1730 47 Salameh M D A., Wiegel J - Effects of detergents on activity, thermostability and aggregation of two alkalithermophilic lipases from Thermosyntropha lipolytica, The Open Biochemistry Journal (2010) 22-28 48 Jonossen I B., Svendson I B - Identification of the reactive sites in two homologous serine prteinase inhibitors isolated from barley, Corlsberg Res Commun 47 (1982) 199203 49 Yang S Q., Wang C I., Gillmor S A., Fletterick R J., Craik C S - Ecotin: a serine protease inhibitor with two distinct and interacting binding sites, J Mol Biol 279 (4) (1998) 945-957 50 Urwin P E., Atkinson H J., Waller D A., Mcpherson M J - Engineered oryzacystatinI expressed in hairy roots confers resistance to Globodera pallida, Plant Journal (1) (1995) 121-131 51 Kiggundu A., Goulet M C., Goulet C., Dubuc J F., Rivard D., Benchabane M., Pepin G., Vyver C V D., Kunert K., Michaud D - Modulating the proteinase inhibitory profile of a plant cystatin by single mutations at positively selected amino acid sites, Plant J 48 (3) (2006) 403–413 52 Koiwa H., D’Urzo M P., Assfalg-Machleidt I., Salzman K Z., Shade R E., Murdock L L., Machleidt W., Bressan R A., Hasegawa P M - Phage display selection of hairpin loop soyacystatin variants that mediate high affinity inhibition of a cysteine proteinase, Plant J 17 (5) (2001) 383–391 53 Issac M., Aknin M., Gauvin-Bialecki A., Voogd N D., Ledoux A., Frederich M., Kashman Y., Carmeli S - Cyclotheonellazoles A–C, Potent Protease Inhibitors from the 420 Protease inhibitors from marine sponge and sponge-associated microorganisms Marine Sponge Theonella aff Swinhoei, J Nat Prod 80 (4) (2017) 1110–1116 54 Fusetani N., Matsunaga S., Matsumoto H., Takebayashi Y - Bioactive marine metabolites 33 Cyclotheonamides, potent thrombin inhibitors, from a marine sponge Theonella sp., J Am Chem Soc 112 (19) (1990) 7053–7054 55 Nakao Y., Matsunaga S., Fusetani N - Three more cyclotheonamides, C, D, and E, potent thrombin inhibitors from the marine sponge Theonella swinhoei, Bioorganic and Medicinal Chemistry (8) (1995) 1115-1122 56 Nakao Y., Oku N., Matsunaga S., Fusetani N - Cyclotheonamides E2 and E3, new potent serine protease inhibitor from the marine sponge of the genus Theonella, J Nat Prod 61 (5) (1998) 667-670 57 Oli S., Abdelmohsen U R., Hentschel U., Schirmeister T - Identification of Plakortide E from the Caribbean Sponge Plakortis halichondroides as a Trypanocidal Protease Inhibitor using Bioactivity-Guided Fractionation, Mar Drugs 12 (5) (2014) 2614-2622 58 Tabares P., Degel B., Schaschke N., Hentschel U., Schirmeister T - Identification of the protease inhibitor miraziridine A in the Red sea sponge Theonella swinhoei, Phcog Res (1) (2012) 63-66 59 Schaschke N - Miraziridine A: natures blueprint towards protease class-spanning inhibitors, Bioorg Med Chem Lett 14 (4) (2004) 855-857 60 Nakao Y., Fujita M., Warabi K., Matsunaga S., Fusetani N - Miraziridine A, a novel cysteine proteae inhibitor from the marine sponge Theonella aff.mirabilis, J Am Chem Soc 122 (42) (2000) 10462-10463 61 Kimura J., Ishizuka E., Nakao Y., Yoshida W Y., Scheuer P J., Kelly –Borges M Isolation of 1-methylherbipoline salts of halisulfate-1 and of suvanine as serine protease inhibitors from a marine sponge, Coscinoderma mathewsi, J Nat Prod 61 (2) (1998) 248-250 62 Carroll A R., Buchanan M S., Edser A., Hyde E., Simpson M., Quinn R J - Dysinosins B−D, Inhibitors of Factor VIIa and Thrombin from the Australian Sponge Lamellodysidea chlorea, J Nat Prod 67 (8) (2004) 1291–1294 63 Nurhayati T., Suptijah P., Suhartono M T., Febrian dan I - Penapisan Inhibitor Protease Yang Dihasilkan Oleh Sponge Asal Kepulauan Seribu, Buletin Teknologi Hasil Perikanan (2) (2004) 45-59 64 de Lira S P., Seleghim M H R., Williams D E., Marion F., Hamill P., Jean F., Andersen R J., Hajdu E., Berlinck R G S - A SARS-coronovirus 3CL protease inhibitor isolated from the marine sponge Axinella cf corrugata: structure elucidation and synthesis, Journal of the Brazilian Chemical Society 18 (2) (2007) 440-443 65 Nakao Y., Masuda A., Matsunaga S., Fusetani N - Pseudotheonamides, Serine Protease Inhibitors from the Marine Sponge Theonella swinhoei, J Am Chem Soc 121 (11) (1999) 2425–2431 66 Craiga K S., Williams D E., Hollander I., Frommer E., Mallon R., Collins K., Wojciechowicz D., Tahir A., Van Soest R., Andersena R J - Novel sesterterpenoid and norsesterterpenoid RCE-protease inhibitors isolated from the marine sponge Hippospongia sp, Tetraedron Letters 43 (27) (2002) 4801-4804 67 Fujimoto Y., Salam K A., Furuta A., Matsuda Y., Fujita O., Tani H., Ikeda M., Kato N., Sakamoto N., Maekawa S., Enomoto N., de Voogd N J., Nakakoshi M., Massayoshi T., 421 Tran Thi Hong, Ton That Huu Dat, Pham Viet Cuong, Nguyen Thi Kim Cuc Sekiguchi Y., Tsuneda S., Akimitsu N., Noda N., Yamashita A., Tanaka J., Moriishi K Inhibition of Both Protease and Helicase Activities of Hepatitis C Virus NS3 by an Ethyl Acetate Extract of Marine Sponge Amphimedon sp, PLoS ONE (11) (2012) e48685 68 Nakao Y., Matsunaga S., Fusetani N - Toxadocial A: A novel thrombin inhibitor from the marine sponge Toxadocia cylindrica, Tetraedron Letters 34 (9) (1993) 1511-1514 69 Meyer M., Guyot M - 5,9,23‐Triacontatrienoic methyl ester, an elastase inhibitor from the marine sponge Chondrilla nucula, Lipids 37 (11) (2003) 1109-1111 70 Fukuhara K., Takada K., Okada S., Matsunaga S - Nazumazoles D-F, Cyclic Pentapeptides That Inhibit Chymotrypsin, from the Marine Sponge Theonella swinhoei, J Nat Prod 79 (6) (2016) 1694-1697 71 Murayama S., Imae Y., Takada K., Kikuchi J., Nakao Y., van Soest R W., Okada S., Matsunaga S - Shishicrellastatins, inhibitors of cathepsin B, from the marine sponge Crella (Yvesia) spinulata, Bioorg Med Chem 19 (22) (2011) 6594-6598 72 Murayama S., Nakao Y., Matsunaga S - Asteropterin, an inhibitor of cathepsin B, from the marine sponge Asteropus simplex, Tetraedron Letters 49 (26) (2008) 4186-4188 73 Dai J., Sorribas A., Yoshida W Y., Kelly M., Williams P G - Xestosaprols from the Indonesian marine sponge Xestospongia sp., J Nat Prod 73 (6) (2010) 1188-1891 74 Fujita M., Nakao Y., Matsunaga S., Seiki M., Itoh Y., van Soest R W M., Fusetani N Ancorinosides B-D, inhibitors of membrane type matrix metalloproteinase (MT1MMP), from the marine sponge Penares sollasi Thiele, Tetrahedron 57 (7) (2001) 12291234 75 Fujita M., Nakao Y., Matsunaga S., Seiki M., Itoh Y., Yamashita J., van Soest R W., Fusetani N - Ageladine A: an antiangiogenic matrixmetalloproteinase inhibitor from the marine sponge Agelas nakamurai, J Am Chem Soc 125 (51) (2003) 15700-15701 76 Shim J S , Lee H S., Shin J., Kwon H J - Psammaplin A, a marine natural product, inhibits aminopeptidase N and suppresses angiogenesis in vitro, Cancer Letters 203 (2) (2004) 163-169 77 Rodríguez-Nieto S., González-Iriarte M., Carmona R., Muñoz-Chápuli R., Medina M A., Quesada A R - Antiangiogenic activity of aeroplysinin-1, a brominated compound isolated from a marine sponge, FASEB Journal 16 (2) (2002) 261-263 78 Mayer A.M.S., Lehmann V.K.B - Marine pharmacology in 1998: marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, anthelmintic, antiplatelet, antiprotozoal, and antiviral activities; with actions on the cardiovascular, endocrine, immune, and nervous systems; and other miscellaneous mechanisms of action, Pharmacologist 42 (2000) 62–69 79 Lee Y K., Lee J H., Lee H K - Microbial symbiosis in marine sponges, J Microbiol 39 (4) (2001) 254–264 80 Calcabrini C., Catanzaro E., Bishayee A., Turrini E., Fimognari C - Marine sponge natural products with anticancer potential: an updated review, Mar Drugs 15 (10) (2017) E310 81 Mayer A M S., Rodríguez A D., Taglialatela-Scafati O., Fusetani N - Marine pharmacology in 2012-2013: marine compounds with antibacterial, antidiabetic, antifungal, anti-inflammatory, antiprotozal, antituberculosis, and antiviral activities; 422 Protease inhibitors from marine sponge and sponge-associated microorganisms affecting the immune and nervous systems, and other miscellaneous mechanisms of action, Mar Drugs 15 (9) (2017) E273 82 Ruocco N., Costantini S., Palumbo F., Costantini M - Marine sponges and bacteria as challenging sources of enzyme inhibitors for pharmacological applications, Mar Drugs 15 (6) (2017) 173 83 Unson M D., Faulkner D.J - Cyanobacterial symbiont biosynthesis of chlorinated metabolites from Dysidea herbacea (Porifera), Experientia 49 (4) (1993) 349-353 84 Fisch K M., Gurgui C., Heycke N., van der Sar S A., Anderson S A., Webb V L., Taudien S., Platzer M., Rubio B K., Robinson S J., Crews P., Piel J - Polyketide assembly lines of uncultivated sponge symbionts from structure-based gene targeting, Nat Chem Biol (7) (2009) 494-501 85 Tabares P., Pimentel-Elardo S M., Schirmeister T., Hünig T., Hentschel U - Antiprotease and immunomodulatory activities of bacteria associated with Caribbean sponges, Mar Biotechnol 13 (5) (2011) 883–892 86 Pimentel-Elardo S M., Buback V., Gulder T A M., Bugni T S., Reppart J., Bringmann G., Ireland C M., Schirmeister T., Hentschel U - New Tetromycin Derivatives with AntiTrypanosomal and Protease Inhibitory Activities, Mar Drugs (10) (2011) 1682-1697 87 Ramadan A U., Szesny M., Othman E M., Schirmeister T., Grond S., Stopper H., Hentschel U - Antioxidant and Anti-Protease Activities of Diazepinomicin from the Sponge-Associated Micromonospora Strain RV115, Mar Drugs 10 (10) (2012) 22082221 88 Wahyudi A T., Qatrunnada, Mubarik N S - Screening and characterization of protease inhibitors from marine bacteria associated with sponge Jaspis sp., HAYATI journal of Biosciences 17 (4) (2010) 173-178 89 Nurhayati T., Suhartono M T., Nuraida L., Poerwanto S B - Karakterisasi Awal Inhibitor Protease dari Bakteri yang Berasosiasi dengan Spons Asal Pulau Panggang, Kepulauan Seribu, HAYATI Journal of Biosciences 13 (2) (2006) 58-64 90 Pimentel-Elardo S M., Grozdanov L., Proksch S., Hentschel U - Diversity of nonribosomal peptide synthetase genes in the microbial metagenomes of marine sponges, Mar Drugs 10 (6) (2012) 1192-1202 91 Cheng-Jian J., Hao Z., Zeng R., Shen P., Li J., Wu B - Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome, Mar Drugs (9) (2011) 1487-1501 423 ... Carmeli S - Cyclotheonellazoles A–C, Potent Protease Inhibitors from the 420 Protease inhibitors from marine sponge and sponge- associated microorganisms Marine Sponge Theonella aff Swinhoei, J Nat Prod... Protease inhibitors from marine sponge and sponge- associated microorganisms against many different proteases For example, cyclotheonellazoles isolated from sponge Theonella inhibited various proteases... summarised protease inhibitors with focusing on their classification, action mechanism, and characters as well as protease inhibitors from marine sponge and spongeassociated microorganisms The marine